Noncovalent Chiral Domino Effect on One-Handed Helix of

stability of a chiral peptide prevailing one-handed helix strongly through the midpoint L-residue. In addition, the N-terminal moiety of a 310-helical...
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Biomacromolecules 2003, 4, 122-128

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Noncovalent Chiral Domino Effect on One-Handed Helix of Nonapeptide Containing a Midpoint L-Residue Yoshihito Inai,* Hisatoshi Komori, Akinori Takasu, and Tadamichi Hirabayashi Department of Environmental Technology and Urban Planning, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Received August 5, 2002; Revised Manuscript Received October 31, 2002

We here clarify whether noncovalent chiral domino effect characterized by the terminal interaction of a helical peptide with a chiral small molecule can alter the helical stability of N-deprotected peptides containing an L-residue covalently incorporated into the inner position. Two nonapeptides consisting of the midpoint L-leucine (1) or L-phenylalanine (2) and the achiral helix-forming residues were employed. NMR and IR spectroscopy and energy calculation indicated that both peptides adopt a 310-helical conformation in chloroform. They strongly preferred a right-handed screw sense because of the presence of the midpoint L-residue. These original right-handed screw senses were retained on addition of chiral Boc-amino acid, but their helical stabilities clearly depended on its added chirality. Here, Boc-L-amino acid stabilizes the original right-handed helix, whereas the corresponding Boc-D-amino acid tends to less stabilize or destabilize it. This tendency was not observed for the corresponding N-Boc-protected peptides 1 and 2, strongly suggesting that the N-terminal amino group is required for controlling the stabilization of the original right-handed helix. Therefore, noncovalent chiral domino effect in peptides 1 and 2 can contribute even to the helical stability of a chiral peptide prevailing one-handed helix strongly through the midpoint L-residue. In addition, the N-terminal moiety of a 310-helical peptide was found to generate chiral discrimination in complexation process with racemic additives. Introduction In general, a biological helix such as peptides and proteins chooses one-handed helix from both left-handed and righthanded ones. This alternative originates from the presence of chiral moieties incorporated into main chain or side chain of a polymer through the covalent bond. Recently, interesting noncovalent chiral inductions have been found in systems of an optically inactive helical polymer with chiral molecule(s).1 Such polymers possess functional groups in the repeating units that can interact with chiral small molecules on their polymer side chains or main chains to lead to the predominant formation of one-handed helix. Moore and coworkers have also proposed a very unique chiral induction that a cylindrical inner space created by helical formation of an achiral backbone captures a chiral molecule to induce a predominantly one-handed helix.1d In contrast to such chiral induction through side-chain or main-chain interactions, we have found another unique noncovalent chiral induction based on one terminus of an optically inactive helical peptide.2 Herein, an N-deprotected achiral nonapeptide, which alone adopts no preferred screw sense of the 310-helix, undergoes the predominance of onehanded helix through the addition of a chiral small carboxylic acid. This chiral stimulus operates on the N-terminal amino group to generate a chiral environment at the N-terminus, in turn leading to continuous chiral amplification along the * To whom correspondence should be addressed. E-mail: inai@ mse.nitech.ac.jp.

single achiral chain. We termed this terminal chiral induction “noncovalent chiral domino effect (NCDE)”. As for chiral induction of achiral peptides, Nielsen and co-workers have already proposed a very elegant system that an achiral peptide nucleic acid (PNA) backbone undergoes chiral duplex formation with the complementary PNA chain containing a chiral residue at the N- or C-terminus,1e-g wherein the chiral induction occurs basically through the side-chain base pairings. Meanwhile, our system might be classified into chiral induction of a single-strand helix through nonbonded interaction with a dissimilar chiral molecule. As exemplified in polyisocyanates,3 oligosilanes,4 and oligopeptides,5 a one-handed helicity of an optically inactive longer chain is induced by a single chiral moiety covalently incorporated into one terminus. Such terminal chiral induction might be classified into “covalent chiral domino effect”, according to our definition. In a comparison between both domino effects, the noncovalent type will enable one to control the original helicity through external chiral stimulus, whereas the covalent type, in fixed conditions such as temperature and solvent, might provide an intrinsic screwsense bias for achiral segment. There have been a great number of outstanding studies on the control of the helical screw sense of biological macromolecules6 or synthetic polymers7 through external stimuli such as pH, light, temperature, solvent, and chiral molecules. In most of these examples, such external stimuli act on a whole polymer molecule, including the main and side chains. On the other hand, the NCDE has another unique interaction

10.1021/bm0256303 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/07/2002

Noncovalent Chiral Domino Effect on Chiral Peptides

manner in which the site-specific action of external stimulus (chiral molecule) upon a terminal moiety of a helical chain can control the original screw sense. In fact, the “tug-ofwar experiment”8 demonstrates that the NCDE also enables one to control the original helicity of an N-deprotected chiral peptide consisting of C-terminal chiral segment and the preceding optically inactive helical segment. Therefore, the NCDE provides novel insights into the nature of chiral intermolecular interactions of a helical peptide with a chiral molecule in peptide and protein science. Meanwhile, there are only few examples for controlling the original helicity of chiral peptides through the NCDE triggered by a small chiral molecule, other than the above peptides containing a C-terminal chiral residue or segment.8 In particular, it is mostly unknown whether the NCDE enables control of the whole helical stability of peptides containing an L-residue covalently incorporated into the inner position, but not into the C-terminus. Such chiral peptides might meet common style of naturally occurring helical peptides, wherein inner L-residue constituents dominate the whole helical screw sense. To clarify this point, we here have adopted two kinds of N-deprotected nonapeptides (Chart 1) containing achiral helicogenic residues of R-aminoisobutyric acid (Aib)9 and R,β-didehydrophenylalanine (∆ZPhe).10 These peptides bear covalently an L-residue (X*) as a single chiral site, leucine (Leu, 1) or phenylalanine (Phe, 2), in the middle position (fifth from each terminus), wherein the chiral center will provide a one-handed helix preferentially. Herein added chiral carboxylic acid will give rise to the NCDE, of which chiral bias spreads over the whole chain, consequently enabling the control of the original helicity. Chart 1: H-(Aib-∆ZPhe)2-X*-(∆ZPhe-Aib)2-OMe where X* ) L-leucine (1) and L-phenylalanine (2)

Experimental Section Materials. All amino acids and coupling reagents were purchased from Tokyo Kasei Co. (Tokyo, Japan) or Kokusan Chemical Works Ltd. (Tokyo, Japan). Boc-amino acid (Boc ) t-butoxycarbonyl) was prepared by a standard procedure with (Boc)2O or was purchased from Kokusan Chemical Works Ltd. Chloroform dried over CaSO4 was distilled onto CaSO4 before use. N,N-Dimethylformamide (DMF) was purified by distillation with ninhydrin under a reduced pressure. Thin-layer chromatography (TLC) was done on precoated silica plates in the following solvent systems: (A) ethyl acetate, (B) methanol, (C) chloroform-methanol (9: 1), and (D) 1-butanol-acetic acid-water (7:2:1). Single spot in the TLC was obtained for each of the final products and their intermediates, as shown below. Synthesis. The pentapeptide Boc-X*-(∆ZPhe-Aib)2-OMe was prepared by ring-opening reaction of Boc-X*-∆ZPhe azlactone with H-Aib-∆ZPhe-Aib-OMe, according to refs 8 and 11. The unprotected H-X*-(∆ZPhe-Aib)2-OMe obtained

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from removal of the Boc group with trifluoroacetic acid (TFA) was coupled with Boc-X*-∆ZPhe azlactone by the same ring-opening reaction to yield the hexapeptide BocAib-∆ZPhe-X*-(∆ZPhe-Aib)2-OMe, according to ref 8. Likewise, the similar stepwise elongation procedure yields the nonapeptide Boc-(Aib-∆ZPhe)2-X*-(∆ZPhe-Aib)2-OMe, which was followed by the Boc-deprotection manner2,8 to yield peptides 1 and 2. The characterization data for the final peptides and the Boc-protected nonapeptides are follows. Boc-(Aib-∆ZPhe)2-L-Leu-(∆ZPhe-Aib)2-OMe. RfA 0.67; B Rf 0.94; RfC 0.67; RfD 0.94. MS (MALDI-TOF) (m/z) [M + Na]+ (calcd for C64H79N9O12Na, 1189.36): found, 1189.21. 200 MHz 1H NMR (δ, in CDCl3): 9.11 + 9.00 + 8.57 + 8.28 (4H, s + s + s + s, NH ∆ZPhe), 8.14 (1H, d, J ) 4.67 Hz, NH Leu(5)), 7.77 + 7.70 (3H, s + s, NH Aib), 7.557.10 (25H, m, NH Aib + 4(CβH + phenyl) ∆ZPhe), 5.26 (1H, s, NH Aib(1)), 4.24 (1H, m, CRH Leu(5)), 3.68 (3H, s, COOCH3), 2.07-1.65 (3H, m, CβH2 + CγH Leu(5)), 1.63 + 1.61 + 1.56 + 1.39 + 1.29 + 1.23 (24H, s + s + s + s + s + s, 8CH3 Aib), 1.42 (9H, s, 3CH3 Boc), 0.88 (6H, m, 2CH3 Leu(5)). FT-IR (cm-1, in chloroform): 1735, 1661, 1623, 1532. FT-IR (cm-1, in KBr): 3290, 1738, 1659, 1627, 1531. Boc-(Aib-∆ZPhe)2-L-Phe-(∆ZPhe-Aib)2-OMe. RfA 0.56; B Rf 0.94; RfC 0.62; RfD 0.94. MS (MALDI-TOF) (m/z) [M + Na]+ (calcd for C64H79N9O12Na, 1223.37): found, 1223.46. 200 MHz 1H NMR (δ, in CDCl3): 9.12 + 8.57 + 8.24 (4H, s + s + s, NH ∆ZPhe), 8.14 (1H, d, J ) 4.67 Hz, NH Phe(5)), 7.78 + 7.67 (2H, s, NH Aib), 7.59-6.90 (25H, m, NH Aib + 4(CβH + phenyl) ∆ZPhe), 5.10 (1H, s, NH Aib(1)), 4.42 (1H, bs, CRH Phe(5)), 3.69 (3H, s, COOCH3), 3.23 (2H, m, CβH2 Phe(5)), 1.65 + 1.62 + 1.60 + 1.59 + 1.57 + 1.36 + 1.27 + 1.24 (24H, s + s + s + s + s + s + s + s, 8CH3 Aib). FT-IR (cm-1, in chloroform): 1734, 1662, 1626, 1531. FT-IR (cm-1, in KBr): 3291, 1739, 1660, 1626, 1530. H-(Aib-∆ZPhe)2-L-Leu-(∆ZPhe-Aib)2-OMe. RfA 0-0.43; B Rf 0.82; RfC 0.59; RfD 0.71. MS (MALDI-TOF) (m/z) [M + Na]+ (calcd for C59H71N9O10Na, 1089.24): found, 1089.39. 600 MHz 1H NMR (δ, in CDCl3): 9.52 (1H, s, NH ∆ZPhe(4)), 8.98 (1H, s, NH ∆ZPhe(6)), 8.06 (1H, s, NH ∆ZPhe(8)), 8.11 (1H, d, J ) 3.46 Hz, NH Leu(5)), 7.79 (1H, s, NH Aib(9)), 7.73 (1H, s, NH Aib(7)), 7.50-7.13 + 6.88 (24H, m + s, 4(CβH + phenyl) ∆ZPhe), 6.88 (1H, s, NH Aib(3)), 4.18 (1H, m, CRH Leu(5)), 3.66 (3H, s, COOCH3), 1.99-1.6 (3H, m, CβH2 + CγH Leu(5)), 1.59 + 1.57 + 1.56 + 1.54 + 1.29 + 1.25 + 1.23 + 1.18 (24H, s + s + s + s + s + s + s + s, 8CH3 Aib), 0.89 (6H, m, 2CH3 Leu(5)). FT-IR (cm-1, in chloroform): 1736, 1661, 1627, 1531. FTIR (cm-1, in KBr): 3290, 1738, 1659, 1627, 1531. In the NOESY spectrum, the relative intensity (%) of NiH-Ni+1H (i - i + 1) cross-peaks on setting the diagonal volume of the ∆ZPhe(4) NH to 100% was as follows: 2.6 (3-4), 2.4 (4-5), 3.0 (5-6), 3.7 (6-7), 2.4 (7-8), and 3.1 (8-9); in other cross-peaks, 1.6 for NH Leu(5)-CRH Leu(5) and 0.09 for CRH Leu(5)-NH ∆ZPhe(6). H-(Aib-∆ZPhe)2-L-Phe-(∆ZPhe-Aib)2-OMe. RfA 0-0.48; RfB 0.85; RfC 0.62; RfD 0.72. MS (MALDI-TOF) (m/z) [M

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+ Na]+ (calcd for C62H69N9O10Na, 1123.26): found, 1123.42. 600 MHz 1H NMR (δ, in CDCl3): 9.59 (1H, s, NH ∆ZPhe(4)), 9.09 (1H, s, NH ∆ZPhe(6)), 8.59 (1H, s, NH ∆ZPhe(8)), 8.09 (1H, d, J ) 2.94 Hz, NH Phe(5)), 7.82 (1H, s, NH Aib(9)), 7.69 (1H, s, NH Aib(7)), 7.52-7.04 + 6.89 (24H, m + s, 4(CβH + phenyl) ∆ZPhe), 6.89 (1H, s, NH Aib(3)), 4.41 (1H, bs, CRH Phe(5)), 3.66 (3H, s, COOCH3), 3.26-3.12 (2H, m, CβH2 Phe(5)), 1.60 + 1.55 + 1.32 + 1.28 + 1.18 + 1.09 (24H, s + s + s + s + s + s, 8CH3 Aib). FT-IR (cm-1, in chloroform): 1736, 1660, 1627, 1530. FT-IR (cm-1, in KBr): 3291, 1739, 1660, 1626, 1530. In the NOESY spectrum, the relative intensity (%) of NiHNi+1H (i - i+1) cross-peaks on setting the diagonal volume of the ∆ZPhe(4) NH to 100% was as follows: 1.9 (3-4), 1.6 (4-5), 2.1 (5-6), 2.3 (6-7), 2.0 (7-8), and 2.4 (8-9); in other cross-peaks, 2.4 for NH Phe(5)-CRH Phe(5) and 0.28 for CRH Phe(5)-NH ∆ZPhe(6). Spectroscopic Measurement. 1H NMR spectra were recorded using Bruker DRX-600 (600 MHz) or DPX-200 (200 MHz) spectrometers at a peptide concentration of 8 mM in CDCl3 or CDCl3/(CD3)2SO at 299 K. All chemical shifts in parts per million (ppm) were determined using tetramethylsilane as an internal standard, and the assignment of NH resonances was made from correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra. NOESY spectra were measured on Bruker DRX-600 (600 MHz) using a Bruker standard pulse program (noesytp)12 with a mixing time of 700 ms, 64 transients per t1, 2 K data points in the t2 domain, and 256 points in the t1 domain. The data processing and analysis were also performed with the XWINNMR software. FT-IR spectra were recorded for chloroform solution containing 1 mM peptide concentration, using a JASCO FT/IR-430 spectrometer. CD and UV spectra were recorded in chloroform on JASCO J-500 (or J-600), and JASCO V-550 spectrometers, respectively. The chloroform was purified by distillation before use. The ∆ZPhe concentration was determined using maximum absorbance around 280 nm (assignable to a ∆ZPhe residue) and its molar extinction coefficient (max ) 1.8 × 104). MALDI-TOF mass spectra of final nonapeptides were acquired on PerSeptive Biosystems Voyager RP in reflectron mode, using anthracene-1,8,9-triol (dithranol) matrix and NaI salt for the sample preparation. For the corresponding N-Bocprotected nonapeptides, 2,5-dihydroxybenzoic acid was used as the matrix. Thin-layer chromatography (TLC) was carried out on precoated silica plates in the following solvent systems: (A) ethyl acetate, (B) methanol, (C) chloroformmethanol (9:1), and (D) 1-butanol-acetic acid-water (7:2:1). Single spot in the TLC and single peak in the GPC were obtained for the final peptides and the Boc-protected peptides. Conformational Energy Calculations. Energy-minimized conformations of peptides 1 and 2 were obtained using the semiempirical molecular orbital (MO) calculation (AM1 method)13 in MOPAC97.13 The minimization with a MOPAC97 keyword of MMOK was carried out for the variables of all bond lengths, bond angles, and torsion angles. An initial conformation of peptides 1 and 2 for the AM1 calculation was obtained from molecular mechanics on the

Inai et al.

Figure 1. Solvent dependence on NH chemical shifts of peptides 1 (A) and 2 (B) in CDCl3/(CD3)2SO mixtures. The ∆ZPhe(2) NH resonance could not be observed because of its broadening.

modified PEPCON;14 all local minima (81)15 of L-Leu or L-Phe residue were employed for the initial conformation for the PEPCON, wherein the remaining achiral residues were set to a standard right-handed 310-helix (φ ) -60.0°, ψ ) -30.0°, ω ) 180°)16 on the basis of the experimental data. Results and Discussion Conformation of Peptides 1 and 2. The solution conformation of peptides 1 and 2 can be expected to adopt a helix, because oligopeptides having -(Aib-∆ZPhe)m- (m ) 2-4) are strongly prone to form a 310-helical structure in solution and in the solid states.2,8,11,17 This prediction, in fact, was evidenced by 1H NMR and FT-IR spectroscopy in solution. In NOESY spectra of both peptides in CDCl3, marked cross-peaks were observed for the neighboring NH pairs [NiH-Ni+1H resonances; NN(i,i+1)] in the segment of Aib(3) to Aib(9), thus indicating the presence of 310-helix or R-helix.18 Herein, the NOE of CRH ∆ZPhe(4) to NH X*(5) [RN(4,5)], which suggests an extended conformation with a ψi value of 120° ( 60°, was considerably small. The observation of NN(i,i+1) NOEs without intense RN(4,5) NOE strongly supports the presence of a helical conformation. Also, the JNH-CRH values of X* residues, 3.7 Hz (Leu, 1) and 3.1 Hz (Phe, 2), correspond to φLeu ) -56° and φPhe ) -49°,19 being typical of a helical conformation. To further specify the helix type, Figure 1 shows the variation in NH chemical shifts of peptides 1 and 2 with concentration of (CD3)2SO in CDCl3.20 Six NH resonances of ∆ZPhe(4) to Aib(9) residues are shielded from solvent because of intramolecular hydrogen bonding, of which the pattern should be assigned to a 310-helix21 supported by successive (i + 3) f i hydrogen bonds starting from NH ∆ZPhe(4) f CO Aib(1). Moreover, the helical conformation was supported by the peak positions of amide I absorption bands of their FT-IR spectra in chloroform: 1661 and 1628 cm-1 for 1 and 1660 and 1627 cm-1 for 2, which can be assigned to saturated amino acid and ∆ZPhe residues incorporated into a helical segment,8,22 respectively. Furthermore, energy minimization of both peptides gave a 310-helical conformation for the peptapeptide segment ∆ZPhe(2)-∆ZPhe(8), as shown in Figure 2: average values for ∆ZPhe(2)-∆ZPhe(8)

Noncovalent Chiral Domino Effect on Chiral Peptides

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Figure 3. CD (top) and UV absorption (bottom) spectra of peptides 1 (solid line) and 2 (dashed line) in chloroform. ∆ and  are expressed with respect to the molar concentration of ∆ZPhe residues.

Figure 2. Side (top) and top (bottom) views23 of energy-minimized conformations of peptides 1 (A) and 2 (B) by the semiempirical MO calculation (AM1 method).13 Both peptides adopt a right-handed 310helical conformation for the heptapeptide segment ∆ZPhe(2)∆ZPhe(8).

residues are 〈φ〉 ) -41.5°, 〈ψ〉 ) -37.1°, and 〈ω〉 ) 179.5° for peptide 1 and 〈φ〉 ) -42.0°, 〈ψ〉 ) -37.6°, and 〈ω〉 ) 179.7° for peptide 2. In both energy-minimized conformations, six NHs of ∆ZPhe(2)-Aib(9) residues participated in successive (i + 3) f i hydrogen bonds starting from NH ∆ZPhe(4) f CO Aib(1), as proposed from the NMR study. Helical Screw Sense of Peptides 1 and 2. The preferred screw sense of peptides 1 and 2 prior to addition of chiral carboxylic acid was investigated by CD spectroscopy. Both peptides in chloroform showed a strong exciton splitting centered at around 280 nm assignable to ∆ZPhe residue (Figure 3). The band around 280 nm has been assigned to charge transfer between the styryl and carbonyl groups.10b (For a detailed CD analysis on ∆ZPhe-containing peptides, see ref 10b.) The split CD sign with a negative peak at longer wavelengths can be assigned to a right-handed screw sense for a 310-helix or R-helix from the exciton chirality method24 and theoretical CD calculation.2,8,11,25 Thus, the central L-Leu or L-Phe residue generates a predominant bias toward a righthanded screw sense for the entire chain. This direction should meet a screw sense preference that is commonly expected for the L-residues. Similar results were found in oligopeptides containing a single L-residue at their inner position of achiral peptide segment, which adopt a right-handed helix on the whole. For example, the terminally blocked hepta- and octapeptides -(Aib)n-L-Leu-(Aib)2- (n ) 4 and 5) were found to adopt a right-handed 310-helix in the crystalline state.26 Also, the terminally blocked pentapeptide -(Aib)2-

Figure 4. CD (top) and UV absorption (bottom) spectra of peptide 1 in chloroform containing (A) Boc-L-Pro-OH or (B) Boc-D-Pro-OH: [1] ) 0.14 mM and [Boc-Pro-OH] ) 0-150 mM. L-Y*-(Aib)2-

(Y* ) L-valine (Val) and CR-methyl L-Val) prefer a right-handed 310-helix in solution.5b Furthermore, an incipient right-handed 310-helix was found in Ac-∆ZPheL-Ala-∆ZPhe-NHMe in the solid state.27 On the other hand, a left-handed helix was found in oligopeptides consisting of achiral segment -(Aib-∆ZPhe)mand an N-terminal or C-terminal L-residue (X*); e.g., BocX*-(Aib-∆ZPhe)m-Aib-OMe5c,11,17a and Boc-(Aib-∆ZPhe)mX*-OMe8,11 (m ) 2 or 4). In the former case, the N-terminal L-residue adopts a semiextended conformation, in turn leading to a left-handed screw sense for the following achiral segment.11,17a For another positional effect, a heptapeptide possessing an L-residue (X*) in the position second from N-terminus, Boc-Aib-X*-(Aib-∆ZPhe)2-Aib-OMe, exhibits both helical screw senses, depending on type of solvent.17b,17c Thus, the shift of an L-residue from N-terminus to an inner position certainly enhances the normal tendency to give a right-handed helix. Noncovalent Domino Effect on Helical Stability. Peptides 1 and 2 alone prefer a right-handed helix in chloroform because of the midpoint L-residue. Figure 4 shows CD spectra of a chloroform solution of peptide 1 in the presence of added chiral carboxylic acid (Boc-Pro-OH, Pro ) proline) with various concentrations. On the addition of Boc-L-Pro-OH (Figure 4A), the split CD sign assignable to a right-handed screw sense is retained, but the split amplitude somewhat

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Inai et al. Table 1: Signs of Splitting Cotton Effects and ∆ Values for Induced CD of Chiral Peptides 1 and 2 with Added Chiral Carboxylic Acida first Cotton effect peptide/X*

added acid

sign

∆/λb

sign

∆/λb

1/L-Leu

none Boc-Gly-OH Boc-DL-Pro-OH Boc-L-Pro-OH Boc-L-Val-OH Boc-L-Leu-OH Boc-L-Ala-OH Boc-L-Phe-OH Boc-D-Pro-OH Boc-D-Val-OH Boc-D-Leu-OH Boc-D-Ala-OH Boc-D-Phe-OH none Boc-Gly-OH Boc-DL-Pro-OH Boc-L-Pro-OH Boc-L-Val-OH Boc-L-Leu-OH Boc-L-Ala-OH Boc-L-Phe-OH Boc-D-Pro-OH Boc-D-Val-OH Boc-D-Leu-OH Boc-D-Ala-OH Boc-D-Phe-OH

-

10.0/302 11.4/302 12.4/302 12.1/303 11.7/302 11.3/303 11.3/303 11.4/302 10.8/301 10.5/302 10.4/301 10.7/301 10.8/302 6.2/301 6.0/300 6.8/302 7.5/302 7.9/303 7.2/303 7.2/302 7.7/303 4.9/301 4.8/300 4.7/300 5.6/301 6.2/301

+ + + + + + + + + + + + + + + + + + + + + + + + + +

20.0/271 22.2/270 23.5/270 24.3/270 24.0/270 23.5/271 23.2/270 c 19.4/270 18.9/270 19.1/271 19.5/269 c 11.9/270 12.4/271 13.6/270 16.4/272 16.7/271 16.5/273 15.2/272 c 8.8/267 9.0/270 8.9/268 10.4/267 c

Figure 5. CD (top) and UV absorption (bottom) spectra of peptide 2 in chloroform containing (A) Boc-L-Pro-OH or (B) Boc-D-Pro-OH: [1] ) 0.14 mM and [Boc-Pro-OH] ) 0-150 mM.

increased: ca. 1.3-fold at [Boc-L-Pro-OH] ) 65 mM. It is obvious that the original right-handed helix can be stabilized by the addition of Boc-L-Pro-OH. On the other hand, the addition of Boc-D-Pro-OH, as shown in Figure 4B, also retains the original right-handed screw sense, and increases the original CD amplitude slightly, similarly to the addition of Boc-L-Pro-OH. However, the CD increase by Boc-L-ProOH is more prominent than that by Boc-D-Pro-OH, strongly implying that the chiral stimulus of Boc-L-Pro-OH stabilizes a right-handed helical structure more effectively than that of Boc-D-Pro-OH. This finding becomes more remarkable in peptide 2 containing the midpoint L-Phe residue, as shown in Figure 5 and Table 1. The addition of Boc-L-amino acid increases the split CD amplitude of peptide 2, in a similar manner as for peptide 1, thus indicating that the original right-handed helix is stabilized by the chiral stimulus of BocL-amino acid. On the other hand, Boc-D-Pro-OH decreases the split CD amplitude markedly, thereby demonstrating that the opposite chiral stimulus destabilizes the original righthanded helix. Similar tendency was observed for addition of other chiral Boc-amino acids (alanine (Ala), Leu, Val, and Phe), as shown in Figures 6 and 7 and Table 1. That is, Boc-L-amino acids tend to more stabilize the original righthanded helix, whereas the original right-handed helix is less stabilized or destabilized by the corresponding Boc-D-amino acids. Strictly speaking, the changes in the split CD amplitude of a right-handed helix should be ascribed to the varying contents of a right-handed helix among all conformers including a left-handed helix and random coils. One might ask at this point what type of conformational transition gives rise to the induced decrease in CD amplitudes, that is, a righthanded to a left-handed helix or a helix to a random coil. To understand the origin of the CD change, we performed the following experiment. When to a pure chloroform solution of peptides 1 and 2 was added 1 vol % of TFA, the original split CD patterns around 280 nm completely disappeared to produce only positive CD signals (see Supporting Information). Herein, the strong acidic nature of TFA breaks the original helix to transform into a random coil structure. Accordingly, the positive CD signals can be assigned to random coil structures. A similar CD assignment was made for terminally blocked peptides possessing a

second Cotton effect

2/L-Phe

a All spectra were recorded using JASCO J-500 in chloroform with [1 or 2] ) 0.14 mM and [Boc-amino acid] ) 100 mM at 18 °C. b ∆ (M-1 cm-1) and λ (nm). c Overlapped with CD signal of Boc-L (or D)-Phe-OH.

Figure 6. CD spectra of peptide 1 in chloroform containing (A) BocL-amino acid (Ala, Leu, Val, Phe, and Pro) or (B) the corresponding Boc-D-amino acid: [1] ) 0.14 mM; [Boc-amino acid] ) 65 mM. The CD spectra of the mixtures containing Boc-L (or D)-Phe-OH are not shown below 272 nm because ofoverlap of the peptide and Boc-L (or D)-Phe-OH signals.

-∆ZPhe-X-∆ZPhe- unit in chloroform-TFA mixtures.10b Meanwhile, the addition of a limited concentration of Bocamino acid, owing to its much weaker acidity than TFA, will not transform the original helix into random coils. Actually, even in pure acetic acid, which has acidity similar to Boc-amino acids, both peptides 1 and 2 still maintain a split-type CD pattern of a right-handed helix (see Supporting Information). Thus, the main origin of the CD change might result from the varying ratios of right-handed to left-handed helices.

Noncovalent Chiral Domino Effect on Chiral Peptides

Figure 7. CD spectra of peptide 2 in chloroform containing (A) BocL-amino acid (Ala, Leu, Val, Phe, and Pro) or (B) the corresponding Boc-D-amino acid: [2] ) 0.14 mM; [Boc-amino acid] ) 65 mM. The CD spectra of the mixtures containing Boc-L (or D)-Phe-OH are not shown below 272 nm because of overlap of the peptide and Boc-L (or D)-Phe-OH signals.

Figure 8. Titration curves of the amplitudes of split CD signals in the complexation of peptide 1 (A) or 2 (B) with Boc-L-Pro-OH (O), Boc-D-Pro-OH (0), Boc-DL-Pro-OH (D/L ) 50/50; 4), or Boc-Gly-OH (2) in chloroform. The dashed line represents the arithmetic mean of the two amplitudes induced by Boc-D-Pro-OH (0) and Boc-L-Pro-OH (O). Titration curves of the amplitudes of split CD signals in the complexation of N-Boc-protected peptide 1 (C) or 2 (D) with Boc-LPro-OH (O), Boc-D-Pro-OH (0), or Boc-Gly-OH (2) in chloroform are also shown: [1] or [2] ) 0.14 mM.

To eliminate the influence of achiral acid-base interaction on CD spectra, the split CD amplitude was plotted against concentrations of Boc-D-Pro-OH, Boc-L-Pro-OH, and achiral Boc-Gly-OH, as shown in Figure 8A,B. In both peptides, Boc-D-Pro-OH destabilizes the original right-handed helix, on the basis of the titration curve of Boc-Gly-OH, whereas Boc-L-Pro-OH stabilizes it markedly. The directions of the helical screw senses induced by Boc-L-Pro-OH and Boc-DPro-OH agree with those observed for the achiral or chiral peptides.2,8 Namely, achiral nonapeptide, H-(Aib-∆ZPhe)4Aib-OMe, generates a right-handed helix through the addition of Boc-L-amino acid.2 Also, chiral peptide containing a C-terminal L-L doublet, H-(Aib-∆ZPhe)4-L-Leu2-OMe, shows a strong bias toward a right-handed helix, which can be stabilized by Boc-L-amino acid but be destabilized by BocD-amino acid.8 N-Boc-protected peptides 1 and 2, Boc-(Aib-∆ZPhe)2-X*(∆ZPhe-Aib)2-OMe, showed a split CD spectrum corre-

Biomacromolecules, Vol. 4, No. 1, 2003 127

sponding to a right-handed screw sense. Although addition of Boc-L-Pro-OH, Boc-D-Pro-OH, or Boc-Gly-OH slightly increased its original CD amplitude, the three titration curves in each peptide (Figure 8C,D) gave essentially the same profile. Obviously, the N-terminal amino group is required for controlling the stabilization of the original right-handed helix. Therefore, the NCDE, characterized by the terminal interaction of a helical peptide with a chiral molecule, operates on peptides 1 and 2 to influence the original helicity. Another interesting phenomenon was observed for the complexation of peptide 1 (or 2) with racemic Boc-Pro-OH, as shown in Figure 8. The CD amplitude induced by BocDL-Pro-OH (D/L ) 50/50) was somewhat larger over the concentration range of Boc-Pro-OH (0-200 mM) than the arithmetic mean (dashed line) of the two amplitudes induced by Boc-D-Pro-OH and Boc-L-Pro-OH. This demonstrates that peptides 1 and 2 adopting a right-handed helix should prefer the binding to Boc-L-Pro-OH rather than to Boc-D-Pro-OH because Boc-L-Pro-OH induces a right-handed helix for achiral peptide H-(Aib-∆ZPhe)4-Aib-OMe.2 A similar tendency was found in the peptide possessing a C-terminal L-L doublet, H-(Aib-∆ZPhe)4-L-Leu2-OMe,8 which adopts a stable right-handed helix. This finding should imply the significant new functionality of a right-handed 310-helix, of which the N-terminal moiety is capable of generating chiral discrimination in its complexation process with racemic additives. While it has been proposed that many helical backbones offer chiral discrimination and recognition,28 our findings seem to be unique in terms of discrimination generated by one terminus of a helical chain. The detailed mechanism is under investigation. In conclusion, the NCDE enables us to influence the original helicity of the chiral peptides that prevail a onehanded helix strongly through the midpoint L-residue. Our current focus is on the NCDE for peptides bearing a chiral residue at the N-terminus. As a preliminary experiment, an N-deprotected H-L-Leu-(∆ZPhe-Aib)4-OMe, which alone adopts a weak bias toward a left-handed helix, was employed. When to a chloroform solution of the peptide (0.13 mM) was added Boc-X-OH (X ) D-Pro, L-Pro, or Gly; 12 mM), the original split CD amplitude increases, thus implying that these added acids all stabilize the original left-handed helix. However, a marked difference in the CD amplitudes (i.e., in degree of stabilization of the left-handed helix) was observed: 7 for the peptide alone, 24 for L-Pro, 36 for Gly, and 44 for D-Pro. Thus, the NCDE seems to be still effective for chiral peptides bearing an N-terminal chiral residue. It should be noted that model peptides composed of Aib or ∆ZPhe residues or both appeal close structural similarities to naturally occurring peptides and proteins that are composed mainly of R-amino acid residues.29 Thus, our findings obtained from noncovalent chiral inductions on achiral R-amino acid sequence might provide novel insights into the nature of helicity inherent in natural peptide backbones. Acknowledgment. We are extremely grateful to Professor Mark M. Green, Herman F. Mark Polymer Research Institute, Polytechnic University, for informing us of valuable literatures on chiral polymers. We also thank M. Eng. K. Tagawa

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for partial support for sample preparation. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan under a grant to Y.I. Supporting Information Available. CD (top) and UV (bottom) spectra of peptides 1 (a) and 2 (b) in chloroform, TFA-chloroform (1/99 v/v), and acetic acid (PDF). This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) For synthetic polymers, see: (a) Yashima, E.; Maeda, K.; Okamoto, Y. Nature 1999, 399, 449. (b) Yashima, E.; Matsushima, T.; Okamoto, Y. J. Am. Chem. Soc. 1997, 119, 6345. (c) Schlitzer, D. S.; Novak, B. M. J. Am. Chem. Soc. 1998, 120, 2196. (d) Prince, R. B.; Barnes, S. A.; Moore, J. S. J. Am. Chem. Soc. 2000, 122, 2758. For peptide nuclear acid backbones, see: (e) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norde´n, B. Nature, 1994, 368, 561. (f) Kozlov, I. A.; Orgel, L. E.; Nielsen, P. E. Angew. Chem., Int. Ed. 2000, 39, 4292. (g) Wittung, P.; Eriksson, M.; Lyng, R.; Nielsen, P. E.; Norde´n, B. J. Am. Chem. Soc. 1995, 117, 10167. (2) Inai, Y.; Tagawa, K.; Takasu, A.; Hirabayashi, T.; Oshikawa, T.; Yamashita, M. J. Am. Chem. Soc. 2000, 122, 11731. (3) (a) Maeda, K.; Okamoto, Y. Polym. J. 1998, 30, 100. (b) Okamoto, Y.; Matsuda, M.; Nakano, T.; Yashima, E. Polym. J. 1993, 25, 391. (4) (a) Obata, K.; Kabuto, C.; Kira, M. J. Am. Chem. Soc. 1997, 119, 11345. (b) Obata, K.; Kira, M. Macromolecules 1998, 31, 4666. (5) (a) Pieroni, O.; Fissi, A.; Pratesi, C.; Temussi, P. A.; Ciardelli, F. J. Am. Chem. Soc. 1991, 113, 6338. (b) Pengo, B.; Formaggio, F.; Crisma, M.; Toniolo, C.; Bonora, G. M.; Broxterman, Q. B.; Kamphuis, J.; Saviano, M.; Iacovino, R.; Rossi, F.; Benedetti, E. J. Chem. Soc., Perkin Trans. 2 1998, 1651. (c) Inai, Y.; Ashitaka, S.; Hirabayashi, T. Polym. J. 1999, 31, 246. (6) (a) Blout, E. R.; Carver, J. P.; Gross, J. J. Am. Chem. Soc. 1963, 85, 644. (b) Overberger, C. G.; David, K.-H. Macromolecules 1972, 5, 373. (c) Watanabe, J.; Okamoto, S.; Satoh, K.; Sakajiri, K.; Furuya, H.; Abe, A. Macromolecules 1996, 29, 7084. (d) Ueno, A.; Takahashi, K.; Anzai, J.; Osa, T. J. Am. Chem. Soc. 1981, 103, 6410. (e) Ciardelli, F.; Pieroni, O.; Fissi, A.; Carlini, C.; Altomare, A. Br. Polym. J. 1989, 21, 97. (f) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984; Chapter 12. (g) Mahadevan, S.; Palaniandavar, M. Chem. Commun. 1996, 114, 2547. (7) (a) Green, M. M.; Peterson, N. C.; Sato, T.; Teramoto, A.; Cook, R.; Lifson, S. Science 1995, 268, 1860. (b) Li, J.; Schuster, G. B.; Cheon, K.-S.; Green, M. M.; Selinger, J. V. J. Am. Chem. Soc. 2000, 122, 2603. (c) Brunsveld, L.; Folmer, B. J. B.; Meijer, E. W.; Sijbesma, R. P. Chem. ReV. 2001, 101, 4071. (d) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. ReV. 2001, 101, 3893. (e) Nakano, T.; Okamoto, Y. Chem. ReV. 2001, 101, 4013. (f) Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M.; Sommerdijk, N. A. J. M. Chem. ReV. 2001, 101, 4039. (g) Lehn, J.-M. Supramolecular Chemistry; VCH: Weinheim, Germany, 1995. (8) Inai, Y.; Ishida, Y.; Tagawa, K.; Takasu, A.; Hirabayashi, T. J. Am. Chem. Soc. 2002, 124, 2466. (9) (a) Prasad, B. V. V.; Balaram, P. CRC Crit. ReV. Biochem. 1984, 16, 307. (b) Benedetti, E.; Bavoso, A.; Di Blasio, B.; Pavone, V.; Pedone, C.; Crisma, M.; Bonora, G. M.; Toniolo, C. J. Am. Chem. Soc. 1982, 104, 2437.

Inai et al. (10) (a) Jain, R.; Chauhan, V. S. Biopolymers 1996, 40, 105. (b) Pieroni, O.; Fissi, A.; Jain, R. M.; Chauhan, V. S. Biopolymers 1996, 38, 97. (11) Inai, Y.; Kurokawa, Y.; Hirabayashi, T. Biopolymers 1999, 49, 551. (12) Bodenhausen, G.; Kogler, H.; Ernst, R. R. J. Magn. Res. 1984, 58, 370. (13) The AM1 method in MOPAC97 was employed: Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J. Am. Chem. Soc. 1985, 107, 3902. For MOPAC97, see: Stewart, J. J. P. MOPAC97; Fujitsu Ltd: Tokyo, Japan, 1998. (14) For PEPCON, see: (a) Momany, F. A.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. A. J. Phys. Chem. 1975, 79, 2361. (b) Beppu, Y. Comput. Chem. 1989, 13, 101. (c) Sisido, M. Peptide Chemistry 1991; Suzuki, A., Ed.; 1992; pp 105-110. For the modified one, see: (d) Inai, Y.; Kurashima, S.; Hirabayashi, T.; Yokota, K. Biopolymers 2000, 53, 484. (e) Inai, Y.; Hirabayashi, T. Biopolymers 2001, 59, 356. (f) Inai, Y.; Oshikawa, T.; Yamashita, M.; Hirabayashi, T.; Kurokawa, Y. Bull. Chem. Soc. Jpn. 2001, 74, 959. (15) Zimmerman, S. S.; Pottle, M. S.; Nemethy, G.; Scheraga, H. A. Macromolecules 1977, 10, 1. (16) (a) Paterson, Y.; Rumsey, S. M.; Benedetti, E.; Nemethy, G.; Scheraga, H. A. J. Am. Chem. Soc., 1981, 103, 2947. (b) Ramachandran, G. N.; Sasisekharan, V. AdV. Protein Chem. 1968, 23, 283. (17) (a) Inai, Y.; Oshikawa, T.; Yamashita, M.; Hirabayashi, T.; Ashitaka, S. J. Chem. Soc., Perkin Trans. 2 2001, 892. (b) Inai, Y.; Kurokawa, Y.; Hirabayashi, T. Macromolecules 1999, 32, 4575. (c) Inai, Y.; Kurokawa, Y.; Kojima, N. J. Chem. Soc., Perkin Trans. 2 2002, 1850. (18) Wu¨thrich, K.; Billeter, M.; Braun, W. J. Mol. Biol. 1984, 180, 715. (19) Pardi, A.; Billeter, M.; Wu¨thrich, K. J. Mol. Biol. 1984, 180, 741. (20) Pitner, T. P.; Urry, D. W. J. Am. Chem. Soc. 1972, 94, 1399. (21) For 310-helical structures, see: Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350 and references therein. (22) (a) Inai, Y.; Sakakura, Y.; Hirabayashi, T. Polym. J. 1998, 30, 828. (b) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541. (23) The molecular graphics were illustrated using the molecular modeling software Butch Software Studio FREE WHEEL for Windows 0.60E for Molecular Modeling Software, Japan, 2001. (24) (a) Harada, N.; Chen, S. L.; Nakanishi, K. J. Am. Chem. Soc. 1975, 97, 5345. For CD analysis of ∆ZPhe-containing peptides, see: (b) Pieroni, O.; Fissi, A.; Jain, R. M.; Chauhan, V. S. Biopolymers 1996, 38, 87. (25) Inai, Y.; Ito, T.; Hirabayashi, T.; Yokota, K. Biopolymers 1993, 33, 1173. (26) Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Toniolo, C.; Bonora, G. M.; Formaggio, F.; Crisma, M. J. Biomol. Struct. Dyn. 1988, 5, 803. (27) Ciajolo, M. R.; Tuzi, A.; Pratesi, C. R.; Fissi, A.; Pieroni, O. Biopolymers 1992, 32, 717. (28) For examples, see: (a) Okamoto, Y.; Yashima, E. Angew. Chem., Int. Ed. 1998, 37, 1020 and references therein. (b) Yashima, E.; Maeda, K.; Yamanaka, T. J. Am. Chem. Soc. 2000, 122, 7813. (c) Yamamoto, C.; Yashima, E.; Okamoto, Y. J. Am. Chem. Soc. 2002, 124, 12583. (d) Higashi, N.; Koga, T.; Niwa, M. Langmuir 2000, 16, 3482. (29) Inai, Y. Recent Research DeVelopments in Macromolecules; Research Signpost: India, 2002; Chapter 2.

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